Methods and kits for the detection of dna

ABSTRACT

The present invention is related to methods and kits for the detection of deoxyribonucleic acid (DNA), in particular pathogenic microbial DNA, which provide more simple and sensitive detection of DNA compared to similar methods and kits known in the art. The method comprises a) contacting the sample with a plurality of metal nanoparticles functionalised with one or more ribonucleic acid (RNA) probes; b) forming a heteroduplex between the target DNA and RNA probe on an RNA-functionalised metal nanoparticle; c) contacting the heteroduplex of target DNA and RNA probe on the RNA-functionalised metal nanoparticle with an enzyme that cleaves RNA in a DNA-RNA heteroduplex thereby releasing the target DNA; d) repeating steps (b) and (c) until all, or substantially all, of the RNA probes on the plurality of RNA-functionalised metal nanoparticles have been cleaved from the metal nanoparticles; and e) aggregating the metal nanoparticles from which all, or substantially all, of the RNA probes have been cleaved, thereby indicating the presence of the target DNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/341,162, filed Nov. 2, 2016, the disclosure of which is incorporatedherein by reference.

TECHNICAL FIELD

This invention relates to methods and kits for the detection of DNA, inparticular pathogenic microbial DNA.

BACKGROUND ART

The on-site and sensitive detection of pathogens is of criticalimportance to the prevention, surveillance and control of infectiousdiseases and their outbreak at the first onset. While conventionaltechniques such as plate culturing, polymerase chain reaction (PCR) andenzyme-linked immunosorbent assay (ELISA) have been used as thepredominant detection workhorses, they are limited by eithertime-consuming procedure, complicated sample pre-treatment, expensiveanalysis and operation, or inability to be implemented atpoint-of-testing. Significant efforts have been made to improve thelimitations associated with conventional techniques. Among these, goldnanoparticles (AuNPs) have emerged as an excellent candidate forbiosensor design owing to their unique properties. For example,colloidal AuNPs exhibit distinct colours and strong absorption bands inthe visible range of the electromagnetic spectrum that are not presentin the bulk metal. This fascinating optical phenomenon of AuNPs isderived from localized surface plasmon resonance (LSPR), a collectiveoscillation of free electrons in tandem with the incoming photonfrequency. This has provided a range of simplified transducingmechanisms for biosensor design, based on assembly, disassembly, orenlargement of the AuNPs which allow scanometric, colorimetric or evennaked-eye determination. Nucleic acid-modified AuNPs have beenincorporated into biological sensing platforms to provide improvedsensitivity, versatility and portability. Remarkably, the nucleic acidfunctionalized AuNPs not only provide further functionalities such asspecific programmable assembly upon hybridization with theircomplementary counterparts, but also allow enzymatic cleavage, ligationand extension reactions for biosensor development.

Toward this end, studies have focused on incorporating nuclease enzymesand deoxyribozymes (DNAzyme) to cleave or link oligonucleotides toinduce a colorimetric response. DNA endonuclease (DNase I),Pb²⁺-dependent RNA-cleaving DNAzyme (DNAzyme 8-17), exonuclease III (ExoIII) and RNAse H have been used successfully for the detection of Pb²⁺,nucleic acids and folate receptor. DNAzyme 8-17, which cleaves the DNAsubstrate with a single RNA linkage in the presence of Pb²⁺, has beenutilised for the detection of metal ions. In a different approach,incorporating the same 8-17 enzyme, cross-linking of enzyme-substrateand subsequent cleavage and dissociation of AuNPs upon the addition oftarget analyte (Pb²⁺) has been reported. Several studies have alsofocused on Exo III enzyme which catalyzes the stepwise removal ofmononucleotides from blunt or recessed 3′-hydroxyl terminus of duplexDNA. A universal platform has been developed for the detection of DNAbased on Exo III signal amplification. Furthermore, Exo III has beenutilized for the colorimetric detection of folate receptor, in which thetarget induced AuNP aggregation. The utilization of Exo III enzyme hasproven highly sensitive due to repeated hybridization and hydrolysisreactions. In a different approach, AuNPs were modified with EcoRIenzyme and a specific, double stranded DNA probe was designed whichcontained an EcoRI recognition site and complementary sticky ends. AuNPaggregation occurred in the presence of the target (magnesium andphosphate ions), resulting in a colorimetric response. Although highlysuccessful, such enzymatic approaches are limited by the need forrestriction binding sites, extensive probe design, and requirement forfurther amplification steps.

Herein, we present innovative sensing methods and kits based on theunique enzymatic activity of endonucleases, such as RNase H, for thedetection of DNA, such as bacterial DNA, at concentrations down tofemtomolar level. Due to the ubiquitous nature and high levels found infood, especially poultry, Campylobacter jejuni was chosen as the targetfor assay development and to exemplify the method of the invention (seeExamples). The exemplified method utilizes RNA-functionalized AuNPswhich form DNA-RNA heteroduplex structures through specifichybridization with target DNA. Once formed, the DNA-RNA heteroduplex issusceptible to RNase H enzymatic cleavage of the RNA probe, allowing DNAto liberate and hybridize with another RNA strand. This continuouslyhappens until all, or substantially all, of the RNA strands are cleaved,leaving the nanoparticles unprotected, or substantially unprotected, andprone to aggregation upon exposure to a high electrolytic medium. Thecurrent invention overcomes previous limitations associated withenzyme-based methods in that it does not require further amplificationsteps. In addition, enzymes such as RNAse H which are not active onsingle stranded DNA or RNA molecules and only catalyze the cleavage ofRNA within a DNA-RNA heteroduplex, do not require specific recognitionsites for enzymatic cleavage. Furthermore, there is greater versatilityand applicability with regard to probe design and thus potential formultiplexing. RNase H has previously been used for the detection of DNAvia RNA cleavage within a DNA-RNA heteroduplex structure and subsequentrelease of a fluorescence dye to generate a fluorescence signal.^([1])That method has a reported limit of detection (LOD) of 10 pM, whichhighlights the ultra-sensitivity of the method of the present inventionwhich can detect target DNA at 1 pM as determined by the naked eye, oreven down to femtomolar level by spectroscopic analysis (see Examples).The fluorescence-based approach mentioned above is further limited bycost due to the synthesis of fluorescein conjugate and requirement forequipment capable of detecting the fluorescence signals. The presentinvention is significantly different from previous reports as itutilizes the plasmonic properties of metal nanoparticles to produce acolorimetric response, in particular gold nanoparticles which produce ared-to-blue colorimetric response, thus the signal can be visiblydetected by the naked eye. In addition, DNA detection can be performedat isothermal conditions in less than three hours. These advantagesprovide a basis for eradicating the need for a thermal cycler,complicated sample preparation, labelled fluorophores, and expensive andcumbersome read-out equipment. Finally, the application of the presentinvention to a food matrix has also been assessed and it is evident thatthe sensitivity and robustness of the assay is conducive for food safetyanalysis.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention provides a method fordetecting a target deoxyribonucleic acid (DNA) in a sample, the methodcomprising:

a) contacting the sample with a plurality of metal nanoparticlesfunctionalised with one or more ribonucleic acid (RNA) probes;

b) forming a heteroduplex between the target DNA and RNA probe on anRNA-functionalised metal nanoparticle;

c) contacting the heteroduplex of target DNA and RNA probe on theRNA-functionalised metal nanoparticle with an enzyme that cleaves RNA ina DNA-RNA heteroduplex thereby releasing the target DNA;

d) repeating steps (b) and (c) until all, or substantially all, of theRNA probes on the plurality of RNA-functionalised metal nanoparticleshave been cleaved from the metal nanoparticles; and

e) aggregating the metal nanoparticles from which all, or substantiallyall, of the RNA probes have been cleaved, thereby indicating thepresence of the target DNA.

Optionally, the metal nanoparticles comprise noble metal nanoparticles.Optionally, the metal nanoparticles consist of noble metalnanoparticles. Optionally, the metal nanoparticles comprise a mixture ofdifferent types of noble metal nanoparticles. Alternatively, the metalnanoparticles comprise the same, or substantially the same, type ofnoble metal nanoparticles, Noble metal nanoparticles show suitablephysicochemical properties, such as ease of functionalization andlocalized surface plasmon resonance, for use in the present invention.As referred to herein, “metals” include alloys of the recited metals.Optionally, the metal nanoparticles are selected from one of more ofgold nanoparticles, silver nanoparticles, platinum nanoparticles, coppernanoparticles, palladium nanoparticles, ruthenium nanoparticles, rhodiumnanoparticles, osmium nanoparticles, and iridium nanoparticles, oralloys of these metals. Optionally, the metal nanoparticles comprisegold nanoparticles. For the purposes of describing the presentinvention, metal nanoparticles in the form of gold nanoparticles will bedescribed and will be exemplified in the Examples. However, it will beappreciated that the skilled person may use other metal nanoparticles inplace of gold nanoparticles as described herein. It will further beunderstood that gold nanoparticles are the preferred nanoparticles foruse in the present invention since gold nanoparticles can be less toxic,less sensitive to oxidation, and provide more vivid colour and colourchanges to allow better naked eye discrimination of aggregation statesof the nanoparticles, than other metal nanoparticles.

Optionally, the gold nanoparticles are functionalised with one or moreRNA probes by conjugating, optionally covalently conjugating, one ormore RNA probes to each metal nanoparticle. Optionally, the metalnanoparticles are functionalised with one or more RNA probes byconjugating, optionally covalently conjugating, one or more RNA probesto each metal nanoparticle via a modification at the 5′ end,alternatively at the 3′ end, of each RNA probe. A suitable modificationis well known to the person skilled in the art and may be, for example,an amine modification, a sulfide modification, a disulfide modification,a thiol modification, or a dithiol modification at the 5′ end,alternatively at the 3′ end, of each RNA probe. Optionally, the metalnanoparticles are functionalised with one or more RNA probes byconjugating one or more amine-modified RNA probes to each metalnanoparticle. In other words, each of the one or more RNA probes areconjugated to the metal nanoparticles through an amine linkage.Optionally, the metal nanoparticles are functionalised with one or moreRNA probes by conjugating one or more sulfide-modified RNA probes toeach metal nanoparticle. In other words, each of the one or more RNAprobes are conjugated to the metal nanoparticles through a sulfidelinkage. Optionally, the metal nanoparticles are functionalised with oneor more RNA probes by conjugating one or more disulfide-modified RNAprobes to each metal nanoparticle. In other words, each of the one ormore RNA probes are conjugated to the metal nanoparticles through adisulfide linkage. Optionally, the metal nanoparticles arefunctionalised with one or more RNA probes by conjugating one or morethiol-modified RNA probes, optionally an alkanethiol-modified RNA probe,to each metal nanoparticle. In other words, each of the one or more RNAprobes are conjugated to the metal nanoparticles through a thiollinkage. Optionally, the metal nanoparticles are functionalised with oneor more RNA probes by conjugating one or more dithiol-modified RNAprobes to each metal nanoparticle. In other words, each of the one ormore RNA probes are conjugated to the metal nanoparticles through adithiol linkage. Optionally, the metal nanoparticles are functionalisedwith one or more RNA probes by conjugating one or more dithiol-modifiedRNA probes to each metal nanoparticle, wherein said one or moredithiol-modified RNA probes are one or more thioctic acid-modified RNAprobes. In other words, each of the one or more RNA probes areconjugated to the metal nanoparticles through a thioctic acid linkage.Optionally, the metal nanoparticles are functionalised with one or moreRNA probes by conjugating one or more thioctic acid-modified RNA probesto each metal nanoparticle via an N-hydroxysuccimidyl ester of thiocticacid at the 5′ end, alternatively at the 3′ end, of the RNA probe.Optionally, the metal nanoparticles are functionalised with one or moreRNA probes by conjugating one or more thioctic acid-modified RNA probesto each metal nanoparticle via an N-hydroxysuccimidyl (NHS) ester ofthioctic acid at the 5′ end, alternatively at the 3′ end, of the RNAprobe. Derivatives of thioctic acid, such as reduced thioctic acid, arealso suitable for use in modifying RNA probes as described herein. Inaddition, other sulphides, disulfides and thiols suitable for use inmodifying RNA probes as described herein include sulfides in the form ofR—S—R, e.g. 3-(Methylthio)-1-propanol), disulfides in the form ofR—S—S—R, e.g. bis(10-carboxydecyl)disulphide), and thiols in the form ofR—SH, e.g. alkyl thiols, wherein R represents a unsubstituted orsubstituted group comprising but not limited to an aliphatic or aromaticalkane, alkene, or other carbon-containing group of atoms including, butnot limited to, ethylene glycol derivatives, and thiolated ethyleneglycol derivatives such [11-(Methylcarbonylthio)undecyl]tetra(ethyleneglycol), which modifications can coordinate strongly onto the metalsurface. Additional thiol modifications include 1-propanethiol,1-butanethiol, 1-pentanethiol, 1-hexanethiol, 1-heptanethiol,1-octanethiol, 1-nonanethiol, 1-decanethiol, 1-undecanethiol,1-dodecanethiol, 1-tetradecanethiol, 1-pentadecanethiol,1-hexadecanethiol, 1-octadecanethiol, 2-ethylhexanethiol,2-methyl-1-propanethiol, 3-methyl-1-butanethiol, butyl3-mercaptopropionate, tert-dodecylmercaptan, and tert-nonylmercaptan.Additional dithiol modifications include 1,2-ethandithiol,1,3-propanedithiol, 1,4-butanedithiol, 2,3-butanedithiol,1,5-pentanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol,1,9-nonanedithiol, tetra(ethylene glycol) dithiol, hexa(ethylene glycol)dithiol, 2,2′-(ethylenedioxy)diethanethiol, and5,5′-bis(mercaptomethyl)-2,2′-bipyridine. Without wishing to be bound bytheory, it is understood that the conjugating, optionally covalentlyconjugating, one or more RNA probes to each metal nanoparticle via amodification as described herein, in particular, by conjugating one ormore thioctic acid-modified RNA probes to each metal nanoparticle via anN-hydroxysuccimidyl (NHS) ester of thioctic acid at the 5′ end of theRNA probe, provides more stable RNA-functionalised metal nanoparticles.As used herein, “stable” means that a majority of the RNA probes remainattached to the metal nanoparticles, and the RNA probes are able tohybridize with a DNA target under suitable conditions for detectingnucleic acids, when stored for up to four weeks at 4° C. in a suitablebuffer, such as TE buffer. TE buffer typically comprises 10 mM Tris,brought to pH 8.0 with HCl, and 1 mM EDTA.

Optionally, the DNA in the sample is single-stranded DNA. Optionally,the sample contains double-stranded DNA and the sample is treated, priorto or at the same time as step (a) of the method of described herein,such that at least some, all, or substantially all, of thedouble-stranded DNA dissociates to single-stranded DNA. It is known inthe art how one may treat a sample containing double-stranded DNA sothat at least some, all, or substantially all, of the double-strandedDNA dissociates to single-stranded DNA. Optionally, the sample may beheated to a suitable temperature such that the double-stranded DNAdissociates to single-stranded DNA. Optionally, the sample may be heatedto a suitable temperature for a suitable period of time, such that thedouble-stranded DNA dissociates to single-stranded DNA. Optionally, thesample may be heated to a suitable temperature wherein the suitabletemperature is ≥80° C., optionally ≥85° C., further optionally ≥90° C.Optionally, the sample may be heated to a suitable temperature for atleast about 30 seconds, optionally at least about 1 minute, furtheroptionally at least about 2 minutes, to dissociate the double-strandedDNA to single-stranded DNA. Optionally, after the sample has been heatedto a suitable temperature, optionally for a suitable period of time,such that the double-stranded DNA dissociates to single-stranded DNA,the sample is maintained at less than about 10° C., optionally at lessthan about 7.5° C., at less than about 5° C., further at less than about4° C., in order to prevent the single-stranded DNA re-hybridising todouble-stranded DNA. Optionally, the sample is maintained at less thanabout 10° C., optionally at less than about 7.5° C., at less than about5° C., further at less than about 4° C., immediately after heating thesample to a suitable temperature, optionally for a suitable period oftime, to dissociate the double-stranded DNA to single-stranded DNA.Optionally, the RNA probe on the RNA-functionalised metal nanoparticleis single-stranded DNA.

Optionally, the RNA probe is of sufficient length to allow the probe tohybridise with the DNA target. The RNA probes useful in the methods andkits disclosed herein may be of varying lengths. Optionally, the RNAprobe is about 5 to about 200, optionally about 5 to about 100,optionally about 5 to about 75, optionally about 10 to about 50, furtheroptionally about 17 to about 40, nucleotides in length. Optionally, theRNA probe, as disclosed herein, comprises a complementarity domain,which domain comprises part of, or the whole, of the nucleotide sequenceof the RNA probe. Optionally, the nucleotide sequence of thecomplementarity domain is sufficiently complementary to a portion of thenucleotide sequence of the target DNA to permit hybridization of the RNAprobe to the target polynucleotide. Optionally, the length of thecomplementarity domain of the RNA probe is ≥10 nucleotides, optionally≥17 nucleotides, and of sufficient length to ensure specifichybridization with the target DNA. Optionally, the length of thecomplementarity domain of the RNA probe is from about 10 to about 100nucleotides, optionally about 17 to about 40 nucleotides. Optionally,the length of the domain of the RNA probe is integer between 10 and 100nucleotides, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, and 100. By “sufficient length” it is meant anoligonucleotide of greater than or equal to 10 nucleotides, optionallygreater than or equal to 17 nucleotides, that is of a length greatenough to provide the intended hybridisation of the RNA probe to the DNAtarget under the appropriate conditions. Optionally, the nucleotidesequence of the complementarity domain of the RNA probe on theRNA-functionalised metal nanoparticle and the nucleotide sequence of atleast a portion of the target DNA is at least about 50% complementary,optionally at least about 60% complementary, optionally at least about70% complementary, optionally at least about 80% complementary,optionally at least about 90% complementary, optionally at least about95% complementary, optionally 100% complementary, to permithybridization of the target DNA and the RNA probe.

Optionally, the RNA probe has a nucleotide sequence comprising 5′-AGGUGU GGA CGA CGU CAA GUC AUC AUG-3′ (SEQ ID NO: 1), or a nucleotidesequence having 50%, optionally 60%, optionally 70%, optionally 80%,optionally 90%, optionally 95%, optionally 100%, sequence similarity tothe sequence 5′-AGG UGU GGA CGA CGU CAA GUC AUC AUG-3′ (SEQ ID NO: 1).Optionally, the RNA probe has a suitable moiety at the 5′ end,optionally at the 3′ end, of the RNA probe to facilitate coupling of theRNA probe to a suitable modification, optionally an amine modification,a sulfide modification, a disulfide modification, a thiol modification,or a dithiol modification, and thus subsequent conjugation to a metalnanoparticle. Optionally, the RNA probe has an amino (NH₂) moiety at the5′ end, optionally at the 3′ end, of the RNA probe. Optionally, the RNAprobe has a moiety comprising an amino (NH₂) group and a C6, C7, C8, C9,C10, C11, C12 or C13-C20 spacer arm at the 5′ end, optionally at the 3′end, of the RNA probe. Optionally, the RNA probe has a nucleotidesequence comprising 5′-Amino-C6-AGG UGU GGA CGA CGU CAA GUC AUC AUG-3′(i.e. an amino-C6 moiety at the 5′ end of SEQ ID NO: 1). Optionally, theRNA probe has a nucleotide sequence consisting of 5′-Amino-C6-AGG UGUGGA CGA CGU CAA GUC AUC AUG-3′ (i.e. an amino-C6 moiety at the 5′ end ofSEQ ID NO: 1).

The amino-C6 moiety, appended to the 5′ PO₃— group in the RNA probe, maybe illustrated as

Optionally, the amino moiety, and optionally the C6, C7, C8, C9, C10,C11, C12 or C13-C20 spacer arm, can be added the 3′-end of the RNA probeusing any suitable means known in the art, such as a 3′-amino-modifiedsolid support. Optionally, the amino moiety, and optionally the C6, C7,C8, C9, C10, C11, C12 or C13-C20 spacer arm, can be added the 5′-end ofthe RNA probe post-synthetically using any suitable means known in theart. Without wishing to be bound by theory, it is understood that, for athioctic acid-modified RNA probe, the amino moiety facilitates couplingof the RNA probe to the NHS group of the thioctic acid-modification, andthus subsequent conjugation to a metal nanoparticle.

Optionally, the metal nanoparticles are colloidal metal nanoparticles.Optionally, prior to functionalization, the metal nanoparticles aresuspended in a suitable buffer. Optionally, prior to functionalization,the metal nanoparticles are suspended in a sodium citrate buffer.Optionally, the metal nanoparticles are reduced and stored in a sodiumcitrate buffer. Optionally, each metal nanoparticle has a shape selectedfrom a sphere, rod, a polygonal rod, rectangular block, cube, tetrapod,and pyramid. Optionally, each metal nanoparticle is in the shape of asphere. Without wishing to be bound by theory, it is understood thatsphere-shaped metal nanoparticles provide for more simple synthesis, RNAfunctionalization, and/or improved localized surface plasmon resonance(LSPR) properties compared to other shapes of metal nanoparticles.Optionally, a non-functionalised metal nanoparticle has a mean diameterof about 1-100 nm, optionally about 10-100 nm, optionally about 10-50nm, optionally about 10-25 nm, optionally about 10-20 nm, furtheroptionally about 17 nm. Optionally, a functionalised metal nanoparticlehas a mean diameter of about 20-200 nm, optionally about 20-100 nm,optionally about 30-60 nm, optionally about 40-50 nm, further optionallyabout 44.4 nm. Optionally, an aggregated mass of metal nanoparticles hasa mean diameter of about 100-1000 nm, optionally about 500-1000 nm,optionally about 750-1000 nm, optionally about 800-900 nm, furtheroptionally about 854 nm.

Optionally, the heteroduplex comprising the target DNA and the RNA probeon the RNA-functionalised metal nanoparticle is formed under conditionssuitable for formation of an RNA-DNA heteroduplex. Optionally, theheteroduplex comprising the target DNA and the RNA probe on theRNA-functionalised metal nanoparticle is formed under suitable bufferconditions, and optionally suitable temperature conditions and/orsuitable lengths of time, for formation of an RNA-DNA heteroduplex.Suitable buffer conditions are known to the person skilled in the art,and can comprise any suitable buffers which condition allow for specifichybridisation of the target DNA and the RNA probe on theRNA-functionalised metal nanoparticle. Optionally, suitable bufferconditions comprise a Tris-HCl buffer, optionally a modified Tris-HCLbuffer. Optionally, the modified Tris-HCL buffer comprises 20 mM Tris(2-Amino-2-(hydroxymethyl)propane-1,3-diol), 40 mM KCl (potassiumchloride), 8 mM MgCl₂ (magnesium chloride), 1 mM DTT(1,4-Dithiothreitol), and, optionally, 30 μM GSH (glutathione), whereinthe pH is adjusted with HCl (hydrochloric acid) to about pH 7.Optionally, once the double-stranded DNA has been dissociated tosingle-stranded DNA, the sample is cooled to a temperature of about 40°C. to about 70° C., optionally about 40° C. to about 60° C., optionallyat a temperature of about 50° C. to 60° C., optionally at a temperatureof about 60° C. Optionally, said cooling occurs over a period of about 1second to about 1 hour, optionally about 30 seconds to about 30 minutes,optionally about 1 minute to about 20 minutes, optionally about 1 minuteto about 10 minutes. Optionally, the heteroduplex comprising the targetDNA and the RNA probe on the RNA-functionalised metal nanoparticle isformed by maintaining the sample at a suitable temperature wherein thesuitable temperature is about 40° C. to about 70° C., optionally about40° C. to about 60° C., optionally about 50° C. to 60° C., optionallyabout 60° C. Optionally, the heteroduplex comprising the target DNA andthe RNA probe on the RNA-functionalised metal nanoparticle is formed bymaintaining the sample at a suitable temperature for about 0.5 hours toabout 2 hours, optionally for about 0.5 hours to about 1.5 hours,optionally for about 1 hour. Optionally, the nucleotide sequence of thecomplementarity domain of the RNA probe on the RNA-functionalised metalnanoparticle and the nucleotide sequence of at least about a portion ofthe target DNA is sufficiently complementary to permit hybridization ofthe target DNA and the RNA probe under conditions suitable for formationof an RNA-DNA heteroduplex, wherein said conditions are as definedabove.

Optionally, the heteroduplex of target DNA and the RNA probe on theRNA-functionalised metal nanoparticle is contacted with an enzyme underconditions suitable for the enzyme to cleave the RNA probe in theheteroduplex thereby releasing the DNA. Optionally, the heteroduplex oftarget DNA and the RNA probe on the RNA-functionalised metalnanoparticle is contacted with an enzyme under suitable bufferconditions, and optionally suitable temperature conditions and/orsuitable lengths of time, for the enzyme to cleave the RNA probe in theheteroduplex thereby releasing the DNA. Suitable buffer conditions areknown to the person skilled in the art, and can comprise any suitablebuffers which allow for the enzyme to cleave the RNA probe in theheteroduplex thereby releasing the DNA. Optionally, suitable bufferconditions comprise a Tris-HCl buffer, optionally a modified Tris-HCLbuffer. Optionally, the modified Tris-HCL buffer comprises 20 mM Tris(2-Amino-2-(hydroxymethyl)propane-1,3-diol), 40 mM KCl (potassiumchloride), 8 mM MgCl₂ (magnesium chloride), 1 mM DTT(1,4-Dithiothreitol), and, optionally, 30 μM GSH (glutathione), whereinthe pH is adjusted with HCl (hydrochloric acid) to about pH 7.Optionally, the heteroduplex of target DNA and the RNA probe on theRNA-functionalised metal nanoparticle is contacted with an enzyme for atime sufficient for the enzyme to cleave the RNA probe in theheteroduplex thereby releasing the DNA. Optionally, the heteroduplex oftarget DNA and the RNA probe on the RNA-functionalised metalnanoparticle is contacted with the enzyme for a suitable time sufficientfor the enzyme to cleave the RNA probe in the heteroduplex, wherein saidsuitable time is at least about 1 minute, optionally at least about 10minutes, optionally at least about 20 minutes, optionally at least about30 minutes, optionally at least about 40 minutes, optionally at leastabout 50 minutes, optionally at least about 1 hours, thereby releasingthe DNA. Optionally, the heteroduplex of target DNA and the RNA probe onthe RNA-functionalised metal nanoparticle is contacted with the enzymeat a temperature suitable to cleave the RNA probe in the heteroduplexthereby releasing the DNA. Optionally, the heteroduplex of target DNAand the RNA probe on the RNA-functionalised metal nanoparticle iscontacted with the enzyme at a temperature of at least about 15° C.,optionally at least about 20° C., optionally at least about 25° C.,optionally at least about 30° C., optionally at least about 37° C.Optionally, the enzyme is an enzyme that cleaves phosphodiester bonds inthe RNA probe in the DNA-RNA heteroduplex. In other words, the enzyme isan enzyme that specifically cleaves the phosphodiester bonds of the RNAoligonucleotide probe when said RNA probe is in a DNA-RNA heteroduplex,i.e. a heteroduplex comprising the target DNA and the RNA probe.Optionally, the enzyme is a ribonuclease enzyme that cleavesphosphodiester bonds in the RNA probe in the DNA-RNA heteroduplex.Optionally, the enzyme is RNase H. Optionally, the final concentrationof enzyme, optionally ribonuclease enzyme, optionally RNase H, used tocleave the RNA probe in the heteroduplex thereby releasing the DNA isabout 0.01 U to 0.1 U, optionally 0.01 U to 0.05 U, optionally about0.02 U. Optionally, in step (c), the heteroduplex of target DNA and theRNA probe on the RNA-functionalised metal nanoparticle is contacted withRNase H at a temperature of about 37° C. for about 1 hour.

Optionally, at least steps (c) and (d) are carried out at substantiallythe same temperature. Optionally, at least steps (c) and (d) are carriedout at substantially the same, and substantially constant, temperature.Optionally, at least steps (c) and (d) are carried out undersubstantially isothermal conditions. Optionally, steps (a) to (e) arecarried out under substantially isothermal conditions.

Optionally, steps (b) and (c) are repeated until substantially all ofthe RNA probes on the RNA-functionalised metal nanoparticles have beencleaved from the metal nanoparticles. Optionally, steps (b) and (c) arerepeated until at least about 50%, optionally at least about 60%,optionally at least about 70%, optionally at least about 80%, optionallyat least about 90%, optionally at least about 95%, optionally at leastabout 96%, optionally at least about 97%, optionally at least about 98%,optionally at least about 99%, of the RNA probes have been cleaved fromthe metal nanoparticles. Optionally, steps (b) and (c) are repeateduntil 100% of the RNA probes have been cleaved from the metalnanoparticles. It is understood that the extent to which the RNA probeson the RNA-functionalised metal nanoparticles have been cleaved from themetal nanoparticles will correlate with the level of stability of thefunctionalised metal nanoparticles, and the extent of aggregation of themetal nanoparticles under increased electrolytic content, such anincreased NaCl content, of a medium containing the metal nanoparticlesand thus colorimetric response.

Optionally, the metal nanoparticles from which all, or substantiallyall, of the RNA probes have been cleaved are aggregated by increasingthe electrolytic content of a medium containing the metal nanoparticles.Optionally, the medium is a medium comprising the sample which comprisedthe target DNA, and optionally in which the functionalised metalparticles contacted the target DNA, and/or optionally in which theenzyme cleaved RNA in a DNA-RNA heteroduplex thereby releasing thetarget DNA. Optionally, the electrolytic content of the medium isincreased by the addition of a salt. Optionally, the electrolyticcontent of the medium is increased by the addition of sodium chloride(NaCl), potassium chloride (KCl), calcium chloride (CaCl₂), magnesiumchloride (MgCl₂), or manganese(II) chloride (MnCl₂) or mixtures thereof,optionally by the addition of NaCl. Optionally, the electrolytic contentof the medium is increased by the addition of a salt, optionally theaddition of NaCl, to a final concentration of at least about 1 M,optionally at least about 2 M.

Optionally, the target DNA in the sample comprises animal, optionallyhuman, DNA. Optionally, the target DNA in the sample comprisesmicrobial, optionally bacterial, DNA. Optionally, the target DNA in thesample comprises pathogenic, optionally pathogenic microbial, DNA.Optionally, the target DNA in the sample comprises Campylobacter jejuniDNA. Optionally, the sample comprises a sample obtained from an animal,optionally a human. Optionally, the sample comprises a bodily fluid,secretion and or cell, such as bodily fluids, secretions, and cells,such as blood, saliva, sweat, hair, mucus, cerebrospinal fluid, urine,and faeces, obtained from an animal, optionally a human. Optionally, thesample comprises a food sample. Optionally, the food sample comprisesmeat, vegetable, grain, or other foodstuff. Optionally, the sample issuspended in a suitable buffer, optionally a Tris-HCl buffer, optionallya modified Tris-HCl buffer as defined above. Optionally, the samplecomprises a food sample comprising, or potentially comprising,pathogenic microbial DNA, optionally Campylobacter jejuni DNA.Optionally, the target DNA in the sample comprises the sequence 5′-CATGAT GAC TTG ACG TCG TCC ACA CCT-3′ (SEQ ID NO: 2), or a sequence having50%, optionally 60%, optionally 70%, optionally 80%, optionally 90%,optionally 95%, optionally 96%, optionally 97%, optionally 98%,optionally 99%, optionally 100%, sequence similarity to the sequence5′-CAT GAT GAC TTG ACG TCG TCC ACA CCT-3′ (SEQ ID NO: 2).

Optionally, the limit of detection (LOD) of the method described hereinis about ≤1 pM, optionally ≤100 fM, optionally ≤40.7 fM, optionally ≤10fM, optionally ≤2.45 fM, optionally ≤1 fM concentration of target DNA inthe sample. Optionally, the limit of detection (LOD) of the methoddescribed herein is 40.7 fM when a colour change as a result ofaggregation of metal nanoparticles is measured by UV-visible lightspectrophotometer. Optionally, the limit of detection (LOD) of themethod described herein is 2.45 fM when an average size of the metalnanoparticles conjugates is measured by dynamic light scattering (DLS).Optionally, the limit of detection (LOD) of the method described herein,when used to detect DNA in a food sample, is 1.2 pM when a colour changeas a result of aggregation of metal nanoparticles is measured byUV-visible light spectrophotometer. Optionally, the limit of detection(LOD) of the method described herein, when used to detect DNA in a foodsample, is 18 fM when an average size of the metal nanoparticlesconjugates is measured by dynamic light scattering (DLS),

Optionally, the target DNA is present in the sample at a concentrationof less than about 1 fM, optionally less than about 10 fM, optionallyless than about 100 fM, optionally less than about 1 pM, optionally lessthan about 10 pM, optionally less than about 100 pM, optionally lessthan about 1 nM, optionally less than about 10 nM, optionally less thanabout 100 nM, optionally less than about 1 M, optionally less than about1 μM, optionally less than about 10 μM, optionally less than about 1 mM.

Optionally, aggregation of the metal nanoparticles causes the metalnanoparticles to change colour thereby indicating the presence of thetarget DNA. Optionally, the metal nanoparticles comprise goldnanoparticles. Optionally, aggregation of the gold nanoparticles causesthe gold nanoparticles to change colour from red to blue therebyindicating the presence of the target DNA strand. Optionally,aggregation of the gold nanoparticles causes the gold nanoparticleschange its light emission wavelength from about 525 nm, corresponding toRNA-functionalised gold nanoparticles, to a wavelength greater than 525nm, optionally to a wavelength of at least about 530 nm, optionally to awavelength of at least about 535 nm, optionally to a wavelength of atleast about 540 nm, optionally to a wavelength of at least about 545 nm,optionally to a wavelength of at least about 550 nm, optionally to awavelength of at least about 555 nm, corresponding to aggregated goldnanoparticles, thereby indicating the presence of the target DNA strand.Optionally, the change of colour of the metal nanoparticles, optionallygold nanoparticles, is detected by the naked eye. Optionally, the changeof colour of the metal nanoparticles, optionally gold nanoparticles, isdetected spectrophotometrically. Optionally, the change of colour of themetal nanoparticles, optionally gold nanoparticles, is detected using aspectrophotometer, optionally an ultra-violet (UV)-visible lightspectrophotometer. Optionally, aggregation of the metal nanoparticles,optionally gold nanoparticles, is detected using transmission electronmicroscopy. Optionally, aggregation of the metal nanoparticles,optionally gold nanoparticles, is detected using a dynamic lightscattering technique.

In a further aspect, the invention provides a kit for detecting a targetDNA in a sample, wherein the kit is used according to the method of theinvention described herein. Optionally, the kit comprises a plurality ofmetal nanoparticles and one or more RNA probes suitable for thedetection of the target DNA. Optionally, the kit comprises a pluralityof metal nanoparticles functionalised with one or more RNA probessuitable for the detection of the target DNA.

Optionally, the metal nanoparticles comprise noble metal nanoparticles.Optionally, the metal nanoparticles consist of noble metalnanoparticles. Optionally, the metal nanoparticles comprise a mixture ofdifferent types of noble metal nanoparticles. Alternatively, the metalnanoparticles comprise the same, or substantially the same, type ofnoble metal nanoparticles. Optionally, the metal nanoparticles areselected from one of more of gold nanoparticles, silver nanoparticles,platinum nanoparticles, copper nanoparticles, palladium nanoparticles,ruthenium nanoparticles, rhodium nanoparticles, osmium nanoparticles,and iridium nanoparticles, or alloys of these metals. Optionally, themetal nanoparticles are gold nanoparticles.

Optionally, the metal nanoparticles are functionalised with one or moreRNA probes, wherein the one or more RNA probes are conjugated,optionally covalently conjugated, to each metal nanoparticle.Optionally, the metal nanoparticles are functionalised with one or moreRNA probes, wherein the one or more RNA probes are conjugated,optionally covalently conjugated, to each metal nanoparticle via amodification at the 5′ end, alternatively at the 3′ end, of the one ormore RNA probes. A suitable modification is well known to the personskilled in the art and may be, for example, an amine modification, asulfide modification, a disulfide modification, a thiol modification, ora dithiol modification at the 5′ end, alternatively at the 3′ end, ofeach RNA probe. Optionally, the metal nanoparticles are functionalisedwith one or more amine-modified RNA probes, wherein the one or moreamine-modified RNA probes are conjugated, optionally covalentlyconjugated, to each metal nanoparticle. In other words, each of the oneor more RNA probes are conjugated to the metal nanoparticles through anamine linkage. Optionally, the metal nanoparticles are functionalisedwith one or more sulfide-modified RNA probes, wherein the one or moresulfide-modified RNA probes are conjugated, optionally covalentlyconjugated, to each metal nanoparticle. In other words, each of the oneor more RNA probes are conjugated to the metal nanoparticles through asulfide linkage. Optionally, the metal nanoparticles are functionalisedwith one or more disulfide-modified RNA probes, wherein the one or moredisulfide-modified RNA probes are conjugated, optionally covalentlyconjugated, to each metal nanoparticle. In other words, each of the oneor more RNA probes are conjugated to the metal nanoparticles through adisulfide linkage. Optionally, the metal nanoparticles arefunctionalised with one or more thiol-modified RNA probes, wherein theone or more thiol-modified RNA probes, optionally alkanethiol-modifiedRNA probes, are conjugated, optionally covalently conjugated, to eachmetal nanoparticle. In other words, each of the one or more RNA probesare conjugated to the metal nanoparticles through a thiol linkage.Optionally, the metal nanoparticles are functionalised with one or moredithiol-modified RNA probes, wherein the one or more dithiol-modifiedRNA probes are conjugated, optionally covalently conjugated, to eachmetal nanoparticle. In other words, each of the one or more RNA probesare conjugated to the metal nanoparticles through a dithiol linkage.Optionally, the metal nanoparticles are functionalised with one or morethioctic acid-modified RNA probes, wherein the one or more thiocticacid-modified RNA probes are conjugated, optionally covalentlyconjugated, to each metal nanoparticle. In other words, each of the oneor more RNA probes are conjugated to the metal nanoparticles through athioctic acid linkage. Optionally, the metal nanoparticles arefunctionalised with one or more thioctic acid-modified RNA probes,wherein the one or more thioctic acid-modified RNA probes are conjugatedto each metal nanoparticle via an N-hydroxysuccimidyl ester of thiocticacid at the 5′ end, alternatively at the 3′ end, of the RNA probes.Optionally, the metal nanoparticles are functionalised with one or morethioctic acid-modified RNA probes, wherein the one or more thiocticacid-modified RNA probes are conjugated to each metal nanoparticle viaan N-hydroxysuccimidyl (NHS) ester of thioctic acid at the 5′ end,alternatively at the 3′ end, of the RNA probes. Derivatives of thiocticacid, such as reduced thioctic acid, are also suitable for use inmodifying RNA probes as described herein. In addition, other sulphides,disulfides and thiols suitable for use in modifying RNA probes asdescribed herein include sulfides in the form of R—S—R, e.g.3-(Methylthio)-1-propanol), disulfides in the form of R—S—S—R, e.g.bis(10-carboxydecyl)disulphide), and thiols in the form of R—SH, e.g.alkyl thiols, wherein R represents a unsubstituted or substituted groupcomprising but not limited to an aliphatic or aromatic alkane, alkene,or other carbon-containing group of atoms including, but not limited to,ethylene glycol derivatives, and thiolated ethylene glycol derivativessuch [11-(Methylcarbonylthio)undecyl]tetra(ethylene glycol), whichmodifications can coordinate strongly onto the metal surface. Additionalthiol modifications include 1-propanethiol, 1-butanethiol,1-pentanethiol, 1-hexanethiol, 1-heptanethiol, 1-octanethiol,1-nonanethiol, 1-decanethiol, 1-undecanethiol, 1-dodecanethiol,1-tetradecanethiol, 1-pentadecanethiol, 1-hexadecanethiol,1-octadecanethiol, 2-ethylhexanethiol, 2-methyl-1-propanethiol,3-methyl-1-butanethiol, butyl 3-mercaptopropionate,tert-dodecylmercaptan, and tert-nonylmercaptan. Additional dithiolmodifications include 1,2-ethandithiol, 1,3-propanedithiol,1,4-butanedithiol, 2,3-butanedithiol, 1,5-pentanedithiol,1,6-hexanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol, tetra(ethyleneglycol) dithiol, hexa(ethylene glycol) dithiol,2,2′-(ethylenedioxy)diethanethiol, and5,5′-bis(mercaptomethyl)-2,2′-bipyridine.

Optionally, the RNA probe or the RNA probe on the RNA-functionalisedmetal nanoparticle is single-stranded DNA.

Optionally, the RNA probe is of sufficient length to allow the probe tohybridise with the DNA target. The RNA probes useful in the methods andkits disclosed herein may be of varying lengths. Optionally, the RNAprobe is about 5 to about 200, optionally about 5 to about 100,optionally about 5 to about 75, optionally about 10 to about 50, furtheroptionally about 17 to about 40, nucleotides in length. Optionally, theRNA probe, as disclosed herein, comprises a complementarity domain,which domain comprises part of, or the whole, of the nucleotide sequenceof the RNA probe. Optionally, the nucleotide sequence of thecomplementarity domain is sufficiently complementary to a portion of thenucleotide sequence of the target DNA to permit hybridization of the RNAprobe to the target polynucleotide. Optionally, the length of thecomplementarity domain of the RNA probe is ≥10 nucleotides, optionally≥17 nucleotides, and of sufficient length to ensure specifichybridization with the target DNA. Optionally, the length of thecomplementarity domain of the RNA probe is from about 10 to about 100nucleotides, optionally about 17 to about 40 nucleotides. Optionally,the length of the domain of the RNA probe is integer between 10 and 100nucleotides, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, and 100. By “sufficient length” it is meant anoligonucleotide of greater than or equal to 10 nucleotides, optionallygreater than or equal to 17 nucleotides, that is of a length greatenough to provide the intended hybridisation of the RNA probe to the DNAtarget under the appropriate conditions. Optionally, the nucleotidesequence of the complementarity domain of the RNA probe on theRNA-functionalised metal nanoparticle and the nucleotide sequence of atleast a portion of the target DNA is at least about 50% complementary,optionally at least about 60% complementary, optionally at least about70% complementary, optionally at least about 80% complementary,optionally at least about 90% complementary, optionally at least about95% complementary, optionally 100% complementary, to permithybridization of the target DNA and the RNA probe.

Optionally, the RNA probe has a nucleotide sequence comprising 5′-AGGUGU GGA CGA CGU CAA GUC AUC AUG-3′ (SEQ ID NO: 1), or a nucleotidesequence having 50%, optionally 60%, optionally 70%, optionally 80%,optionally 90%, optionally 95%, optionally 100%, sequence similarity tothe sequence 5′-AGG UGU GGA CGA CGU CAA GUC AUC AUG-3′ (SEQ ID NO: 1).Optionally, the RNA probe has a suitable moiety at the 5′ end,optionally at the 3′ end, of the RNA probe to facilitate coupling of theRNA probe to a suitable modification, optionally an amine modification,a sulfide modification, a disulfide modification, a thiol modification,or a dithiol modification, and thus subsequent conjugation to a metalnanoparticle. Optionally, the RNA probe has an amino (NH₂) moiety at the5′ end, optionally at the 3′ end, of the RNA probes. Optionally, the RNAprobe has a moiety comprising an amino (NH₂) group and a C6, C7, C8, C9,C10, C11, C12 or C13-C20 spacer arm at the 5′ end, optionally at the 3′end, of the RNA probes. Optionally, the RNA probe has a nucleotidesequence comprising 5′-Amino-C6-AGG UGU GGA CGA CGU CAA GUC AUC AUG-3′(i.e. an amino-C6 moiety at the 5′ end of SEQ ID NO: 1). Optionally, theRNA probe has a nucleotide sequence consisting of 5′-Amino-C6-AGG UGUGGA CGA CGU CAA GUC AUC AUG-3′ (i.e. an amino-C6 moiety at the 5′ end ofSEQ ID NO: 1).

The amino-C6 moiety, appended to the 5′ PO₃— group in the RNA probes,may be illustrated as

Optionally, the amino moiety, and optionally the C6, C7, C8, C9, C10,C11, C12 or C13-C20 spacer arm, can be added the 3′-end of the RNA probeusing any suitable means known in the art, such as a 3′-amino-modifiedsolid support. Optionally, the amino moiety, and optionally the C6, C7,C8, C9, C10, C11, C12 or C13-C20 spacer arm, can be added the 5′-end ofthe RNA probe post-synthetically using any suitable means known in theart.

Optionally, the metal nanoparticles are colloidal metal nanoparticles.Optionally, prior to functionalization, the metal nanoparticles aresuspended in a suitable buffer. Optionally, prior to functionalization,the metal nanoparticles are suspended in a sodium citrate buffer.Optionally, the metal nanoparticles are reduced and stored in a sodiumcitrate buffer. Optionally, each metal nanoparticle has a shape selectedfrom a sphere, rod, a polygonal rod, rectangular block, cube, tetrapod,and pyramid. Optionally, each metal nanoparticle is in the shape of asphere. Optionally, a non-functionalised metal nanoparticle has a meandiameter of about 1-100 nm, optionally about 10-100 nm, optionally about10-50 nm, optionally about 10-25 nm, optionally about 10-20 nm, furtheroptionally about 17 nm. Optionally, a functionalised metal nanoparticlehas a mean diameter of about 20-200 nm, optionally about 20-100 nm,optionally about 30-60 nm, optionally about 40-50 nm, further optionallyabout 44.4 nm. Optionally, an aggregated mass of metal nanoparticles hasa mean diameter of about 100-1000 nm, optionally about 500-1000 nm,optionally about 750-1000 nm, optionally about 800-900 nm, furtheroptionally about 854 nm.

Optionally, the target DNA to be detected by the kit comprises animal,optionally human, DNA. Optionally, the target DNA to be detected by thekit comprises microbial, optionally bacterial, DNA. Optionally, thetarget DNA to be detected by the kit comprises pathogenic, optionallypathogenic microbial, DNA. Optionally, the target DNA to be detected bythe kit comprises Campylobacter jejuni DNA. Optionally, the target DNAto be detected by the kit is comprised in a sample obtained from ananimal, optionally a human. Optionally, the sample comprises a bodilyfluid, secretion and or cell, such as bodily fluids, secretions, andcells, such as blood, saliva, sweat, hair, mucus, cerebrospinal fluid,urine, and faeces, obtained from an animal, optionally a human.Optionally, the target DNA to be detected by the kit is comprised in afood sample. Optionally, the food sample comprises a meat-basedfoodstuff such as chicken, vegetable-based foodstuff, and/or grain-basedfoodstuff. Optionally, the kit comprises a suitable buffer forsuspending the sample, optionally the suitable buffer is a Tris-HClbuffer, optionally a modified Tris-HCl buffer as defined above.Optionally, the target DNA to be detected by the kit is comprised in afood sample comprising, or potentially comprising, pathogenic microbialDNA, optionally Campylobacter jejuni DNA. Optionally, the target DNA tobe detected by the kit comprises the sequence 5′-CAT GAT GAC TTG ACG TCGTCC ACA CCT-3′ (SEQ ID NO: 2), or a sequence having 50%, optionally 60%,optionally 70%, optionally 80%, optionally 90%, optionally 95%,optionally 96%, optionally 97%, optionally 98%, optionally 99%,optionally 100%, sequence similarity to the sequence 5′-CAT GAT GAC TTGACG TCG TCC ACA CCT-3′ (SEQ ID NO: 2).

Optionally, the limit of detection (LOD) of the kit, optionally whereinthe kit used according to the method described herein, is about ≤1 pM,optionally ≤100 fM, optionally ≤40.7 fM, optionally ≤10 fM, optionally≤2.45 fM, optionally ≤1 fM concentration of target DNA in the sample,Optionally, the limit of detection (LOD) of the kit, optionally whereinthe kit used according to the method described herein, is 40.7 fM when acolour change as a result of aggregation of metal nanoparticles ismeasured by UV-visible light spectrophotometer. Optionally, the limit ofdetection (LOD) of the kit, optionally wherein the kit used according tothe method described herein, is 2.45 fM when an average size of themetal nanoparticles conjugates is measured by dynamic light scattering(DLS). Optionally, the limit of detection (LOD) of the kit, optionallywherein the kit used according to the method described herein, when usedto detect DNA in a food sample is 1.2 pM when a colour change as aresult of aggregation of metal nanoparticles is measured by UV-visiblelight spectrophotometer. Optionally, the limit of detection (LOM) of thekit, optionally wherein the kit used according to the method describedherein, when used to detect DNA in a food sample is 18 fM when anaverage size of the metal nanoparticles conjugates is measured bydynamic light scattering (DLS).

Optionally, the target DNA is present in a sample to be tested by thekit at a concentration of less than about 1 fM, optionally less thanabout 10 fM, optionally less than about 100 fM, optionally less thanabout 1 pM, optionally less than about 10 pM, optionally less than about100 pM, optionally less than about 1 nM, optionally less than about 10nM, optionally less than about 100 nM, optionally less than about 1 μM,optionally less than about 10 μM, optionally less than about 100 μM,optionally less than about 1 mM.

Optionally, the kit further comprises an enzyme that cleaves RNA in aDNA-RNA heteroduplex. Optionally, the kit further comprises an enzymethat cleaves phosphodiester bonds in the RNA probe when the RNA probe isin a DNA-RNA heteroduplex. Optionally, the kit further comprises aribonuclease enzyme that cleaves phosphodiester bonds in the RNA probewhen the RNA probe is in a DNA-RNA heteroduplex. Optionally, the enzymeis RNase H.

Optionally, the kit comprises a salt. Optionally, the kit comprisesNaCl, KCl, CaCl₂, MgCl₂, or MnCl₂, or mixtures thereof. Optionally, thesalt is a solution ofNaCl, KCl, CaCl₂, MgCl₂, or MnCl₂, or mixturesthereof, wherein optionally the salt solution has a concentration ofabout 1 to about 10 M, optionally about 1 to about 10 M, optionallyabout 4 M. Optionally, the salt is a solution of NaCl, whereinoptionally the solution of NaCl has a concentration of about 1 to about10 M, optionally about 1 to about 10 M, optionally about 4 M.

Optionally, the kit further comprises instructions for using the kit.Optionally, the kit further comprises instructions for using the kitaccording to the method for detecting a target DNA in a sample asdescribed herein.

By “about”, as used herein, it is meant that the recited value may beprecisely the recited value, optionally+10% of the recited value,further optionally+20% of the recited value.

By “some”, as used herein, it is meant that the quantity of feature isat least about 20%, optionally at least about 30%, optionally at leastabout 40%, optionally at least about 50%, of the total quantity of thatfeature.

By “substantially all”, as used herein, it is meant that the quantity offeature is at least greater that about 50%, optionally at least about60%, optionally at least about 70%, optionally at least about 80%,optionally at least about 90%, optionally at least about 95%, optionallyat least about 96%, optionally at least about 97%, optionally at leastabout 98%, optionally at least about 99%, of the total quantity of thatfeature.

By “immediately”, as used herein, it is meant that a method step isperformed subsequent to a preceding method step and within at leastabout 60 seconds, optionally at least about 30 seconds, optionally atleast about 20 seconds, optionally at least about 10 seconds, optionallyat least about 1 seconds, optionally at least about 1 second, of thepreceding method step.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Embodiments of the present invention will now be described, by way ofexample, with reference to the accompanying drawings, in which:

FIG. 1 depicts the overall scheme of the method of the invention anddemonstrates the colorimetric detection of target DNA based on DNA-RNAhybridization and enzyme controlled cleavage and aggregation of goldnanoparticles (AuNPs) upon the addition of NaCl. (A) In the presence oftarget DNA, DNA-RNA hybridization occurs which initiates RNAse H enzymecleavage of RNA within the heteroduplex structure. The target DNArecycles until all of the RNA is cleaved, allowing for subsequent AuNPaggregation in the presence of NaCl. (B) In the absence of target DNA,no hybridization occurs, thus there is no heteroduplex structure to acton and the RNAse H enzyme is inactive. Therefore, the particles remainstable upon the addition of NaCl.

FIG. 2 depicts transmission electron microscopy (TEM) images andphotographs of the colorimetric response of AuNPs in the (A) absence and(B) presence of target DNA (1 μM) post assay conditions. Scale bar=100nm.

FIG. 3 depicts the results of analysis of AuNP-RNA conjugates in thepresence of increasing concentrations of target DNA. (A) Arepresentative colour photograph showing a visual colour change from redto blue or transparent with increasing DNA concentrations. (B) UV-visabsorption spectra demonstrating a red shift towards longer wavelengths(525 nm to 555 nm) in the presence of increasing target DNAconcentrations. (C) Wavelength shift of the LSPR peak as a function oftarget DNA concentration (n=3). Inset: the linear relationship betweentarget DNA concentration and maximum wavelength shift (R²=0.93). (D)Hydrodynamic size distribution of AuNPs measured by dynamic lightscattering (DLS) technique. (E) Linear relationship between target DNAconcentration and average increase in AuNP size derived from DLSanalysis (R²=0.98).

FIG. 4 depicts the results of experiments demonstrating assayspecificity. (A) DLS measurements demonstrating the hydrodynamic sizedistribution of AuNP-RNA post assay conditions. From top to bottom:RNAse H enzyme and 0 μM target DNA; no RNAse H enzyme and 10 μM targetDNA; RNAse H enzyme and 1 μM target DNA; RNAse H enzyme and 10 μMnon-complementary DNA sequence 1; and RNAse H enzyme and 10 μMnon-complementary DNA sequence 2. (B) and (C) are the correspondingcolour photograph and maximum wavelength shift of LSPR peak underdifferent conditions (n=3), respectively.

FIG. 5 depicts (A) the wavelength shift of the LSPR peak as a functionof target DNA concentration analyzed in chicken matrix, demonstratingthe sensitivity of the assay. Inset is the linear relationship betweentarget DNA concentration spiked in chicken matrix and maximum wavelengthshift (R²=0.96), and (B) the linear relationship between target DNAprepared in chicken matrix and normalized, average increase innanoparticle size determined by dynamic light scattering technique (DLS)(R²=0.98).

FIG. 6 depicts how RNAse H enzyme and gold nanoparticles (AuNPs) aresuccessfully utilized in the development of a biological sensor for thedetection of nucleic acids at a highly sensitive limit of detection of2.45 fM. AuNPs are functionalized with a specific RNA probe. In thepresence of target DNA, RNAse H enzyme catalyses the cleavage of RNAwithin and RNA-DNA heteroduplex structure, leading to gold nanoparticleaggregation upon addition of NaCl.

FIG. 7 depicts the stability of RNA-AuNP conjugates (A) and barenanoparticles (B) following additional of different concentrations ofNaCl (zero to 2.0 M), further demonstrating successful functionalisationand determination of assay conditions.

FIG. 8 depicts the results of gold nanoparticle (AuNP) characterization.The figures illustrate transmission electron microscopy (TEM)characterization of AuNPs produced via the citrate reduction/Turkevichmethod (A) and size distribution analysis of the particles using ImageJsoftware (B).

FIG. 9 depicts the functionalisation of AuNPs with RNA. The graphdemonstrates the wavelength spectrum (230-800 nm) of bare-AuNP (d=17±3nm, red curve) with a maximum absorbance peak at 520 nm (red arrow), andRNA in buffer alone demonstrating a peak absorbance at 260 nm (blackcurve, black arrow), and the RNA-AuNP conjugates (blue curve). Thisresult was used to confirm successful functionalisation of RNA onto theAuNP surface as demonstrated by an absorbance peak at 525 nm (LSPR) and260 nm (RNA) post centrifuging to remove unbound RNA. Controlexperiments were carried out to provide further illustration that RNA iscleaved by RNAse H enzyme and subsequent addition of NaCl inducesaggregation. The assay was conducted as stated in methods section,subsection Colorimetric detection ofpathogenic bacterial DNA prior toNaCl addition. Post incubation with the endonuclease enzyme, the samplescontaining 0 and M target DNA was centrifuged at 13,000 g for 10minutes, decanted and suspended in L of ultrapure H₂O. UV-Vismeasurements were carried out (230-800 nm) prior to the addition of NaCl(final effective concentration of 2 M) and compared to the fullwavelength spectrum post addition. The results show a diminished peak at260 nm (olive curve), and a broadened plasmonic peak at 556 nm when the2 M NaCl is added (orange curve). This suggests that the enzyme hascleaved the RNA from the surface of the AuNP, resulting in theunprotected AuNPs. In contrast, the RNA absorbance peak 260 nm stillpersists, and no broadened plasmonic peak is observed for the zero DNAsample (magenta curve).

DETAILED DESCRIPTION

Embodiments of the present invention will now be described withreference to the following non-limiting examples:

Methods

Materials:

NH₂ RNA probe (5′-Amino-C6-AGG UGU GGA CGA CGU CAA GUC AUC AUG-3′) (SEQID NO: 1), complementary DNA sequence (5′-CAT GAT GAC TTG ACG TCG TCCACA CCT-3′) (SEQ ID NO: 2), non-complementary DNA sequence 1 (5′-CCA ACCCCC CAG AAA GAA-3′) (SEQ ID NO: 3) and non-complementary sequence 2(5′-TCT ATT GGT AAA ACT TAC GCT GCA AGT AAA GCC GAA GGT CAC-3′) (SEQ IDNO: 4) were purchased from Eurofins Genomics (Ebersburg, Germany). RNAseH enzyme was purchased from Takara Bio (France) and thioctic acid NHSester from Link Technology Ltd. (UK). Sodium citrate tribasic dehydrate(HOC(COONa)(CH₂COONa)₂.2H₂O), gold (III) chloride (HAuCl₄), dimethylsulfoxide (DMSO) ((CH₃)₂SO), Tween20, sodium chloride (NaCl), sodiumcarbonate (Na₂CO₃), sodium bicarbonate (Na₂HCO₃), triethylammoniumacetate buffer (TEAA), sodium phosphate (NaH₂PO₄ and Na₂HPO₄),glutathione (GSH), Tris-EDTA buffer (TE), Tris-HCl (NH₂C(CH₂OH)₃),hydrochloric acid (HCl), potassium chloride (KCl), magnesium chloride(MgCl₂) and dithiothreitol (DTT) were purchased from Sigma-Aldrich (UK).All reagents were prepared in RNAse free water (Sigma, UK). NAP-5 columnwas obtained from GE Healthcare (UK). Syringe filters (0.22 μm) werepurchased from Merck Millipore (Germany).

Gold Nanoparticle Synthesis:

In a typical experiment, 25 mM HAuCl₄ was dissolved in 150 mL dH₂O andheated to reflux under constant stirring (t=˜10 minutes). 1 mL 2.4 mM ofsodium citrate was quickly injected and the solution was removed fromthe heat upon the colour changed from translucent yellow to wine red.The pH of the solution was adjusted to 6.5 using HCl.

RNA Preparation:

Prior to functionalization, the RNA was modified with thiotic acid atthe 5′ end. The dried RNA was incubated with thiotic acid/80 mM DMSO (30μL) and Na₂CO₃/NaHCO₃ (75 μL) overnight at room temperature. Themodified RNA was desalted using a NAP-5 column in TEAA buffer (0.1 M).The concentration was determined using Nanodrop 8000 (Thermoscientific,UK).

Gold Nanoparticle Functionalization:

Gold nanoparticles were functionalized with RNA. Typically, 300 μL of 30μM modified-RNA was added to 1 mL AuNP solution. The solution wasincubated overnight (t=−16 hours) at room temperature and stabilitychecked with 4 M NaCl (2 M). To improve the orientation of the RNAconjugated onto the surface of the AuNP, the salt concentration wasslowly increased. Firstly, phosphate buffer (60 mM) was diluted to afinal concentration of 10 mM. NaCl was added in small increments (0.05M) each hour, over six hours (0.3 M). The conjugate was incubatedovernight (t=−16 hours) at room temperature. Finally, the conjugate wascentrifuged twice at 13,000 g for 1 hour to remove unbound RNA andre-suspended in TE buffer (stored at 4° C.).

Determination of AuNP-RNA Stability:

In order to ensure the colloidal stability of the RNA-functionalizedAuNPs at high salt concentrations, the conjugates were exposed todifferent concentrations of NaCl (FIG. 7). The functionalized particlesexhibit excellent stability at 2 M NaCl, with no wavelength shift noted.The bare-AuNPs immediately aggregated at 0.5 M NaCl, due to the largescreening effect of NaCl, causing a red-shift to longer wavelengths onthe absorbance spectrum (λ_(max) shift >200 nm). This stability can beattributed to electrostatic repulsion or steric exclusion caused by RNAon the AuNP surface. From this analysis, successful functionalisationwas confirmed and 2 M NaCl was set as the highest concentration toinduce aggregation of AuNPs over varying stabilities.

Colorimetric Detection of Pathogenic Bacterial DNA:

Prior to analysis, the AuNP conjugate was centrifuged (13,000 g for 30min) and re-suspended in a modified Tris-HCl buffer (20 mM Tris, 40 mMKCl, 8 mM MgCl₂ and 1 mM DTT). 30 μM GSH was also added to this bufferdue to its role in aiding RNAse H enzyme activity. In a typicalexperiment, 20 μL of AuNP-RNA was added into an Eppendorf tube with 10μL of target DNA or control (modified Tris-buffer) and 1.5 μL ofTween20. The sample was heated to 90° C. for 2 minutes, cooled slowly to60° C. and incubated at 60° C. for 1 hour. 0.06 U of RNAse H enzyme,prepared in modified Tris-buffer, was added to a final concentration of0.02 U. The sample was then incubated for 1 hour at 37° C. To induceaggregation, NaCl was added to a final, effective concentration of 2 M.

Preparation of DNA in Chicken Matrix:

50 g of skinless chicken meat (breast) was shaken for 2 minutes in 100mM Tris-HCl (pH 7). The subsequent matrix was filtered and diluted 1/100in modified Tris-HCl buffer (20 mM Tris, 40 mM KCl, 8 mM MgCl₂ and 1 mMDTT). This matrix was then used to prepare a 10-fold dilution of targetDNA ranging from 0 to 10 μM concentration.

Analysis Instrumentation:

All Ultraviolet-visible spectrophotometry measurements were carried outusing a Cary 60 spectrophotometer (Agilent Technologies, USA). AuNP sizeanalysis was carried out using a Zetasizer NanoZS (Malvern, UK).Transmission electron microscopy (TEM) characterization was acquiredusing a Phillips CM100 (Phillips, USA) operated at 100 kV.

Results & Discussion

Gold nanoparticles (AuNPs) (17±3 nm, FIG. 8) were first synthesized by amethod reported previously with minor alterations^([2]) and exhibited atypical UV-vis absorbance band at 520 nm. Subsequently, asingle-stranded RNA probe (5′-Amino-C6-AGG UGU GGA CGA CGU CAA GUC AUCAUG-3′) was designed to recognize a DNA fragment of Campylobacterjejuni, (NCTC 11168=ATCC 700819 chromosome, 5′-CAT GAT GAC TTG ACG TCGTCC ACA CCT-3′). The RNA probe was successfully crafted onto the AuNPvia an N-hydroxysuccimidyl (NHS) ester of thioctic acid. The AuNP-RNAconjugate exhibits a deep red colour, and absorbance peaks at 525 nm and260 nm (FIG. 9), which represents the typical optical absorption ofAuNPs and RNA, respectively. The shift in LSPR peak from 520 nm (barenanoparticles), to 525 nm with the AuNP-RNA conjugates furtherdemonstrates the successful functionalisation. The RNA-functionalizedAuNPs prepared by this method show excellent stability under highelectrolytic conditions (zero to 2 M NaCl) (FIG. 7). In the presence oftarget Campylobacter jejuni DNA, hybridization occurs with RNAfunctionalized onto the AuNP surface. The subsequent DNA-RNAheteroduplex becomes a target for cleavage of the RNA probe via RNase Henzyme, allowing the DNA to liberate and hybridize with another RNAstrand (FIG. 1, overall scheme). This happens isothermally anditeratively until all of the RNA probes are cleaved, leaving thenanoparticles denuded. The addition of 2 M NaCl causes the denudednanoparticles to aggregate in solution, initiating a colour change fromred to blue. The aggregation state was confirmed in FIG. 2 by thetransmission electron microscopy (TEM) analysis of RNA-functionalizedAuNPs under assay conditions in the absence and presence (1 μM) oftarget DNA. The distinct colour change generated from the assay can bedetected by the unaided eye, or by simple spectroscopic analysis.

To test the hypothesis of the assay, a 10-fold dilution of target DNAranging from 0 to 10 μM concentration was analyzed. FIG. 3A demonstratesthe visible colour change of AuNP-RNA solution from red to blue or grey,with increasing target DNA concentration. The lowest target DNAconcentration easily determined by the naked eye was 10⁻⁶ μM, as seen bya colour change from red to light blue, caused by a decrease ininterparticle distance. More quantitatively, UV-vis absorptionmeasurements were performed to determine the LSPR shift as a function oftarget DNA concentration (FIG. 3B). The results demonstrate a shift inwavelength from a target DNA concentration of 10 fM, which continues toincrease to 10 M (FIG. 3C). A good linear relationship between the LSPRshift and target DNA concentration (FIG. 3C, inset) could be obtainedfor a range between 10 fM and 10 μM of target DNA (R²=0.93). The limitof detection (LOD) is defined as the lowest DNA concentration with aresponse three times greater than the standard deviation (SD) of theblank sample. Owing to the excellent stability of the AuNP-RNAconjugate, the SD value of the zero concentration for ten measurements(n=10) was as small as 1.9 nm, thus the LOD of the current assay is 40.7fM of pathogenic bacterial DNA. The LOD is about two orders of magnitudemore sensitive in comparison to a similar method utilizingfluorescein^([1]) and other AuNP-functionalized based assays for thedetection of DNA.^([1,3,4])

During the UV-vis analysis, we observed that the aggregated AuNPs hadlatched on to the wall of the Eppendorf tube or settled down to thebottom of the tube, potentially causing only free and partiallyaggregated particles to be measured in solution. Thus, it is evidentthat the LSPR shift is not as predominant when compared to thecolorimetric response as seen in the colour photographs (FIG. 3B).Therefore, to accurately reflect the aggregation states of the AuNPs,dynamic light scattering (DLS) measurements were carried out todetermine the average size of the AuNP-conjugates in the presencevarying target DNA concentrations. DLS data is displayed as theintegrated value of three measurements derived from one sample. Thepolydispersity index (PDI) for each sample is also displayed whichindicates the variation of nanoparticle size within a distribution. ThePDI is calculated from the distribution width and mean, giving anoverall indication of the non-uniformity of particles within a sample.The DLS results show an average increase in AuNP size, from 44.4 nm(±1.2 nm) with the zero concentration sample to 854.0 nm (±36.0 nm) at 1M target DNA concentration, which demonstrates increased aggregationwith increasing target DNA concentration (FIG. 3D). Furthermore, thesize distribution charts show a shift in average size from onepopulation, between 10-100 nm to a second, increasing population (100 to1000 nm). This large size distribution can be explained by the highlysensitive nature of DLS analysis which is capable of determining thetrue state of particles in a given media, compared to TEM which onlymeasures the solid state (nanoparticles are dried on grid subsequent toanalysis). Furthermore, DLS measures the hydrodynamic diameter of theparticle, which in this case includes the metallic core and RNAfunctionalised onto the surface, thus it is obvious that one should seethis wide distribution. The PDI data demonstrates broad polydispersionwhich increases from 0 μM to 10⁻³ μM target DNA concentration, where theindex value decreases. This indicates a wide size distribution at lowertarget DNA concentrations, with narrower size distributions at higherconcentrations. This coincides with the distribution data whichindicates that there are a greater number of larger or aggregatedparticles at a high target DNA concentration, in contrast with lowertarget DNA concentrations which demonstrate a larger size frequency overseveral distribution sizes (i.e. 10-100 nm and 100-1000 nm). FIG. 3Edemonstrates the linear fitting of AuNP size increase as a function oftarget DNA concentration (R²=0.98). This data provides a dynamic rangeof between 1 fM and 100 pM of target DNA. From these we can determine aLOD of 2.45 fM of target DNA as there is some degree of AuNPaggregation, which is not visible to the naked-eye or detectable usingUV-vis spectrophotometry. A baseline has been included in FIG. 3E toremove background signal as determined by specificity and selectivityanalysis.

Control experiments were carried out with RNAse H enzyme removed todetermine if nanoparticle aggregation was fully attributed to theenzymatic cleavage of RNA (target DNA concentration was fixed at 10 μM).In the absence of target DNA but in the presence of RNAse H enzyme, theaverage AuNP size was determined to be 44.4±1.2 nm (FIG. 4A) with novisible change in colorimetric response (FIG. 4B) and no shift in LSPRpeak (maximum absorbance peak at 525 nm; FIG. 4C). In the absence ofRNAse H but in the presence of 10 μM target DNA, the hybridization ofthe RNA-DNA resulted in an increase in the average size of thenucleotide-AuNP complex of 24.8 nm as compared to the zero (no target)sample (FIG. 4A). The UV-vis results further demonstrate that theremoval of RNAse H enzyme results in almost no change in the LSPR peakshift. This result confirms that the addition of RNAse H enzyme isessential in controlling aggregation of the AuNPs through cleavage offunctionalised RNA.

Control experiments were also carried out with two non-complementary DNAsequences at a concentration of 10 μM. The DLS results demonstrate aslight increase in particle complex size with a maximum value of 9.6 nm(FIG. 4A), attributing to the background noise of the assay (alsodenoted as the baseline on FIG. 3E). The colour images in FIG. 4B alongwith the UV-vis results (FIG. 4C) demonstrate no visual or spectroscopicchange in colour or LSPR peak under the test experiments. The highspecificity of the assay can be credited to the specific DNA-RNArecognition and hybridisation, as well as subsequent highly selectiveRNAse H enzyme cleavage of the RNA in the heteroduplex.

The applicability of the assay was tested by preparing a 10-folddilution of target DNA ranging from 0 to 10 μM in chicken matrix. FIG.5A shows the maximum LSPR shift, as a function of target DNA in chickenmatrix. The results demonstrate good linearity (R²=0.96) spanning from10 fM and 10 M. In the complex environment, the spiked target DNA wasidentified at concentration as low as 1.2 pM based on the LSPR analysis.It was noted that the LOD had increased two orders of magnitude incomparison to buffer conditions and there was an overall decrease inmaximum LSPR shift (15 nm), which might be attributable to interferencesof the enzymatic reaction within the sample. Furthermore, parallel DLSmeasurements were carried out to determine the aggregation states ofAuNPs post analysis in chicken matrix. FIG. 5B demonstrates the linearcorrelation between normalized average size (nm) and DNA concentration(μM). The linear range of the assay was determined to be between 10 fMand 1 μM, with a lowest detectable limit of around 100 fM. Thus, theassay shows good application to food analysis.

CONCLUSION

In conclusion, we have presented a highly sensitive and selective methodfor the detection of DNA, based on endonuclease controlled aggregationof plasmonic AuNPs. RNAse H enzymatic cleavage in combination withDNA-RNA hybridization provides a highly specific and ultra-sensitiveassay, which can detect 1 pM target DNA concentration visibly or down tofemtomolar level by spectroscopic techniques (40.7 and 2.45 fM asmeasured by UV-vis and dynamic light scattering (DLS), respectively).The detection capabilities within a food matrix show good sensitivity(1.2 pM and 18 fM as analyzed by UV-vis and DLS, respectively). Inaddition to the ultra-high sensitivity, the total analysis time of theassay is less than 3 hours, thus demonstrating its practicality for foodanalysis. The versatility of probe design and enzyme cleavage offers abroad range of potential applications for the detection of DNA. Futurework will focus on further applications of the method and potentialmultiplexing capabilities for real clinical and veterinary samples.

REFERENCES

-   [1] J. Kim, R. Estabrook, G. Braun, B. Lee, N. Reich, Chem. Commun.    (Camb), 2007, 42, 4342-4344;-   [2] J. Turkevich, P. C. Stevenson, J. Hillier, Discuss. Faraday Soc.    1951, 11, 55-75;-   [3] L. Cui, G. Ke, W. Y. Zhang, C. J. Yang, Biosensors and    Bioelectronics, 2011, 5, 2796-2800;-   [4] Q. Fan, J. Zhao, H. Li, L. Zhu, G. Li, Biosensors and    Bioelectronics, 2012, 1, 211-215.

The invention is not limited to the embodiments described herein but canbe amended or modified without departing from the scope of the presentinvention.

1. A method for detecting a target deoxyribonucleic acid (DNA) in asample, the method comprising: a) contacting the sample with a pluralityof metal nanoparticles functionalised with one or more ribonucleic acid(RNA) probes; b) forming a heteroduplex between the target DNA and RNAprobe on an RNA-functionalised metal nanoparticle; c) contacting theheteroduplex of target DNA and RNA probe on the RNA-functionalised metalnanoparticle with an enzyme that cleaves RNA in a DNA-RNA heteroduplexthereby releasing the target DNA; d) repeating steps (b) and (c) untilall, or substantially all, of the RNA probes on the plurality ofRNA-functionalised metal nanoparticles have been cleaved from the metalnanoparticles; and e) aggregating the metal nanoparticles from whichall, or substantially all, of the RNA probes have been cleaved, whereinthe aggregating is performed in the presence of a salt in a finalconcentration of from about 0.5 to 10M, thereby indicating the presenceof the target DNA, and wherein limit of the detection of said method isabout ≤1 pM.
 2. The method of claim 1, wherein the metal nanoparticlescomprise noble metal nanoparticles.
 3. The method of claim 2, whereinthe metal nanoparticles are gold nanoparticles.
 4. The method of claim1, wherein the metal nanoparticles are functionalised with one or moreRNA probes by conjugating the one or more RNA probes to each metalnanoparticle via a modification at the 5′ end or the 3′ end of each RNAprobe.
 5. The method of claim 4, wherein the modification is an aminemodification, a sulfide modification, a disulphide modification, a thiolmodification, or a dithiol modification.
 6. The method of claim 5,wherein the dithiol modification is a thioctic acid modification.
 7. Themethod of claim 1, wherein the sample contains double-stranded DNA andthe sample is treated, prior to or at the same time as step (a), suchthat at least some of the double-stranded DNA dissociates tosingle-stranded DNA.
 8. The method of claim 1, wherein, in step (b), theheteroduplex comprising the target DNA and the RNA probe on theRNA-functionalised metal nanoparticle is formed by maintaining thesample at a temperature of about 40° C. to about 70° C., optionally forabout 0.5 hours to about 2 hours.
 9. The method of claim 1, wherein theenzyme is an enzyme that cleaves phosphodiester bonds in the RNA probein the heteroduplex.
 10. The method of claim 9, wherein the enzyme is aribonuclease enzyme, optionally wherein the enzyme is RNase H.
 11. Themethod of claim 1, wherein at least steps (c) and (d) are carried outunder substantially isothermal conditions.
 12. The method of claim 1,wherein the salt is sodium chloride (NaCl), potassium chloride (KCl),calcium chloride (CaCl₂), magnesium chloride (MgCl₂), Manganese chloride(MnCl₂), or a mixture thereof.
 13. The method of claim 1, wherein theaggregation of the metal nanoparticles causes the metal nanoparticles tochange colour thereby indicating the presence of the target DNA.
 14. Themethod of claim 13, wherein the metal nanoparticles are goldnanoparticles and the aggregation of the gold nanoparticles causes thelight emission wavelength of the gold particles to shift from about 525nm to a wavelength of greater than about 525 nm thereby indicating thepresence of the target DNA.
 15. The method of claim 13, wherein thechange of colour of the nanoparticles is detected by the naked eye,spectrophotometrically, transmission electron microscopy or a dynamiclight scattering technique.
 16. A kit for detecting a target DNA in asample, wherein the kit is used according to the method of claim
 1. 17.The kit of claim 16, wherein the kit comprises a plurality of metalnanoparticles and one or more RNA probes for functionalising the metalnanoparticles and which are suitable for the detection of the targetDNA, wherein optionally the kit further comprises an enzyme that cleavesphosphodiester bonds in the RNA probe in a DNA-RNA heteroduplex.
 18. Thekit of claim 16, wherein the kit comprises gold nanoparticlesfunctionalised with one or more RNA probes suitable for the detection ofthe target DNA, wherein optionally the kit further comprises an enzymethat cleaves phosphodiester bonds in the RNA probe in a DNA-RNAheteroduplex.
 19. The method of claim 1, wherein the limit of detectionof the method is about ≤1 pM as determined by the naked eye.
 20. Themethod of claim 1, wherein the limit of detection of the method is about≤40.7 fM as measured by UV-visible light spectrophotometry.
 21. Themethod of claim 1, wherein the limit of detection of the method is about≤2.45 fM as measured by dynamic light scattering (DLS).